引物延伸预扩增在β-地中海贫血产前基因诊断中的应用研究

Investigation into the application of primer extension preamplification to prenatal genetic diagnosis of β-thalassaemia

目的探讨孕妇外周血中单个胎儿有核红细胞(NRBC)经引物延伸预扩增(PEP)后在β-地中海贫血产前基因诊断中应用的可行性。方法以2004年7月至2005年6月在广西医科大学第一附属医院行产前诊断,夫妇均为轻型β-地中海贫血,孕龄9～34周的28例孕妇为研究对象。显微操作获取母血中NRBC,PEP对单个NRBC进行全基因组扩增,短串联重复序列(STR)鉴定所取细胞的来源。证实为胎儿细胞的PEP产物作为模板进行β-珠蛋白基因扩增,反向点杂交确定胎儿β-珠蛋白基因型。结果每例发现NRBC4～13个,约43.6%的NRBC来源于胎儿。单个NRBC经PEP后STR及β-珠蛋白基因扩增成功率分别为100.0%和90.8%。经反向点杂交可鉴定胎儿β-珠蛋白基因点突变类型,与传统的产前诊断结果相比较,符合率为85.7%,误诊率14.3%。结论母血中单个胎儿NRBC经PEP后可以进行产前基因诊断,不仅可以应用于β-地中海贫血,也可应用于其它遗传病,是一条可供尝试的无创性产前诊断途径。

Objective To investigate the feasibility of β-thalassaemia non-invasive prenatal genetic diagnosis using a single fetal nucleated erythrocyte （NRBC） from maternal blood after primer extension preamplification. Methods From July 2004 to June 2005, 28 couples at risk for transmitting beta-thalassaemia major were investigated ,and blood samples were taken from pregnant women at 9 - 34 gestational weeks in the First Affiliated Hospital of Guangxi Medical University. NRBCs from maternal blood were collected by micromanipulation. The whole genome of a single NRBC was amplified by primer extension preamplification （ PEP）, and then each single NRBC was determined to be fetal or maternal in origin by short tandem repeat （STR） after further amplification of this gene. Once a single NRBC had been identified as being of fetal origin, the β-globin gene fragment was amplified by PCR using small aliquots of the PEP product, the β-thalassaemia mutations was analyzed by reverse dot blot（RDB）. Results NRBCs were found in all of 28 pregnant women at a range of 4 to 13 per 5ml venous blood,and about 43. 6% of NRBCs were determined to be fetal in origin. After PEP, single NRBCs were successfully amplified in 100. 0% of STR PCR and 90. 8% of β-globin gene fragment PCR respectively. The β-thalassaemia mutations of fetal were determined by RDB. The coincidence was 85.7% compared with the result of traditional prenatal diagnosis, misdiagnosis rate being 14. 3%. Conclusion After PEP, prenatal diagnosis can be accomplished by using a single fetal NRBC from the maternal circulation. This method provides a feasible approach for non-invasive prenatal genetic diagnosis for β-thalassaemia and other genetic diseases, including autosomal and X-linked diseases.